GB2150559A - Novel L-proline producing microorganisms and their use - Google Patents

Abstract

L-proline production is improved when a strain of microorganisms resistant to at least two different proline analogues is cultured. An improved strain of Brevibacterium ammoniagenes, designated ATCC No. 39101, has been isolated by selective culture in the presence first of L-azetidine-2-carboxylic acid then 3,4-dehydro-DL-proline, which has an altered genetic structure with respect to the L-proline feedback mechanism, and has been found to produce L-proline in commercial quantities.

Description

SPECIFICATION Novel L-proline producing microorganisms and their use Production of L-proline (referred to herein as "proline") and other amino acids via fermentation has been the subject of considerable research. Microorganisms belonging to the genera Arthrobacter, Brevibacterium, Corynebacterium, Microbacterium and others require a certain amount of proline in order to survive and will produce their own proline. Such proline production is not necessarily dependent on a lack of proline in the medium, i.e., some microorganisms may always produce some quantities of proline. Nonetheless, it is believed thatthemicrobial production of proline is controlled, at least in part, by certain feedback mechanisms such that the microorganisms usually will cease or reduce manufacture of proline when sufficient quantities are available to it.

When an analogue of proline is present, the microorganisms generally will recognize it and utilize it as proline, thus triggering the feedback mechanism to shut down or slow proline production. In the absence of either environmental or internally-produced proline, the microorganisms will die off. Certain mutants are insensitive or resistant to proline and proline analogues. The feedback mechanism of these mutants is not triggered by either proline or proline analogues. Consequently, these mutants will continue to produce more proline than they need.

The patent and technical literature contains numerous references describing proline analogue-resistance as a means of selecting microorganisms which will overproduce proline. For example, Japanese Patent Publication No. 55-148096 describes a process for production of proline by cultivating microorganisms belonging to the genus Corynebacterium, Arthrobacter, Brevibacterium or Microbacterium that are resistant to structural proline analogues such as 3,4-dehydroproline, hydroxyproline, azetidine-2-carboxylic acid, proline hydroxamate and D-proline. Similarly, U.S. 4,244,409, issued to Nakamori et al., discloses a method for producing proline, using mutants which belong to the genus Brevibacterium, Corynebacterium or Microbacterium and which are resistant to DL-3,4-dehydroproline.The proline analogue-resistant microorganisms typically are obtained by exposing known proline producers or known glumatic acid producers to a mutagen and then screening for mutants which will produce proline in the presence of the proline analogue.

Summary of the invention It has been found that unique microorganisms characterized by double proline analogue-resistance are useful for the production of proline in commercial quantities. Specifically, a strain of Brevibacterium ammonia genes has been identified which will produce significantly more proline than either the non-resistant parent strain or a single analogue-resistant intermediate strain. The new strain is resistant to both L-azetidine-2-carboxylic acid ("ACA") and 3,4-dehydro-DL-proline ("DHP"). The proline produced by cultivating microorganisms of this new strain may be separated readily from the fermentation broth by standard methods.

It is an object of this invention to provide a novel microorganism capable of enhanced proline production in commercial quantities.

Another object is to provide a microorganism in which the genetic structure has been altered in such a manner as to render the microorganism resistant to multiple proline analogues.

Moreover, it is intended that the new strain be useful in standard commercial fermentation processes or improvements thereof.

Detailed description and examples This invention is directed to new strains of microorganisms whose altered genetic structure causes or allows the microorganisms to be resistant to two structural proline analogues, ACA and DHP. Consequently, the microorganisms will produce excessive quantities of proline when cultivated under aerobic conditions.

As one embodiment, a new strain was derived from a culture of Brevibacterium ammoniagenes obtained from the American Type Culture Collection under ATCC No. 13746.

Brevibacterium ammonia genes ATCC 13746 is a known glutamic acid producer which was found, in a preliminary screening, to produce small quantities of proline (less than one milligram per milliliter). It was desired to obtain, by resistance to multiple structural proline analogues, a mutant strain which would produce significantly elevated levels of proline. The method of selection involved exposing the ATCC 13746 strain to ACA and searching for a spontaneous mutant with ACA resistance. This intermediate ACA-resistant mutant was exposed to DHP and a mutant with the desired characteristic - multiple analogue resistance was found.

A Nutrient Agar (commercially available from Difco) slant of ATCC 13746 was washed with sterile de-ionized water. A 0.2 ml aliquot of the resulting cell suspension was spread onto a Nutrient Agar (Difco) plate containing 10 mg/ml of the proline analogue ACA. This plate was incubated at 300C for 3 days. Due to the presence of ACA in the culture medium, growth of ATCC 13746 prototrophs was suppressed and only ACA-resistant microorganisms were capable of growth. The largest of the individual resistant colonies which grew on the ACA plate were transferred to a fresh plate of Nutrient Agar (Difco) without ACA. This plate was incubated until the ACA-resistant colonies had grown, approximately 3 days.

One of these ACA-resistant clones was found to produce elevated levels of proline when grown in Proline Medium C, the contents of which are as follows: Ingredient Quantity (per liter de-ionized water) Glucose 50.0 g NH4CI 5.0 g Urea 5.0 g KH2PO4 0.5 g K2HFO4 0.5 g MgSO4 7 H2O 0.5 g FeSO4 .7 H2O 0.02 g MnSO4 4 H2O 0.02 g ZnSO4 7 H2O 0.01 g Biotin 100.0 yg Thiamine-HCI 1.0 mg The pH of the medium was adjusted to neutrality, about 6.8, with sodium hydroxide, and the medium was autoclaved for 12 minutes at 112 C.

The ACA-resistant clone was grown in 50 ml of Proline Medium C in a 250 ml indented Erlenmeyer flask for 3 days at 30"C and at 300 RPM. The broth was harvested, centrifuged at 4000 RPM and filtered through a .22 micron millipore filter. The proline titer was determined by the Technicon AutoAnalyzer II method using isatin dye. The procedure followed was to heat 1% isatin dye in isopropanol with the sample for 1 minute at 80'C, and then determine the absorption at 590 nm. It was found that the ACA-resistant clone produced 8.5 mgiml proline.

This intermediate ACA-resistant mutant was inoculated onto a slant of Nutrient Agar (Difco) and incubated for 4 days at 30 C. The slant was washed with 5.0 ml sterile de-ionized water. A 0.2 ml aliquot of the resulting cell suspension was spread onto a Nutrient Agar (Difco) plate which contained 2 mg/ml DHP. This plate was incubated at 30 C for 3 days.

One of the colonies which grew on this plate - resistant to both ACA and DHP - was transferred to a fresh plate of Nutrient Agar (Difco) which contained neither ACA nor DHP. This strain was then grown in Proline Medium C10S, the contents of which are as follows: Ingredient Quantity (per liter de-ionized water) Glucose 100.0 g NH4CI 5.0 g Urea 5.0 g KH2PO4 0.5 g K2HPO4 0.5 g MgSO4 . 7 H2O 0.5 g FeSO4.7H2O 0.02 g MnSO4.4H2O 0.02 g ZnSO4.7H2O 0.01 g Bacto-Soytone (Difco) 0.3 g Biotin 100.0 g Thiamine-HCI 1.0 mg The pH of the medium was adjusted to neutrality, about 6.8, with sodium hydroxide, and the medium was autoclaved for 12 minutes at 112 C.

One of the ACA and DHP resistant clones was grown for 3 days in 50 ml of Proline Medium C10S in a 250 ml indented Erlenmeyerflask. The proline titer was determined by isatin dye assay, as described above. The clone was found to produce proline at levels of 19.6 mg/ml.

A lyophilized culture of this newly developed ACA- and DHP-resistant strain of Brevibacterium ammoniagenes has been deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockvilie, Maryland 30852, U.S.A. on April 16th 1982 with no restrictions as to availability to the public upon issuance of this patent. The culture has been designated ATCC No. 39101.

An alternative fermentation using ATCC 39101 for the production of proline was done using the following media: Seed Medium Ingredient Quantity Glucose 5.0gum NaCI 5.0 gm Yeast extract 10.0 gm Peptone 10.0gm The ingredients were mixed in de-ionized water to one liter, the pH adjusted to 7.0 with sodium hydroxide, and the medium was autoclaved for 15 minutes at 112"C.

Production Medium Ingredient Quantity' Urea 5.0 gm NH4CI 5.0 gm K2HPO4 1.Ogm MgSO4.7H2O 0.5 7 0.Sgm Soy Isolate (Sheffield Co.) 1.0 gm FeSO47H2O 1.0 7 lOmI2 MnSO4H2O 2.0 ml3 ZnSO47H2O 1.0 7 1.0ml4 Biotin 1.0 ml5 Thiamine-HCI 1.0 mI6 CaCO3 50.0 gm Glucose7 1. The addition of glucose should be taken into account when determining the total volume of de-ionized water.

2. Stock solution: 20.0 gm/l FeSO4 .7 7 H20 3. Stock solution: 6.1 gm/l MnSo4 H2O 4. Stocksolution:10.0gm/lZnSO4 7H2O 5. Stock solution: 0.1 gm/l Biotin 6. Stock solution: 1.0 gm/I thiamine-HCl 7. Stock solution: 70% glucose. Autoclave at 1 10'C for 15 minutes. Add 7.0 ml/flask before inoculating.

The first ten ingredients were mixed with de-ionized water to one liter, the pH adjusted to 6.8 with sulfuric acid and the CaCO3 added. The medium was autoclaved at 121"C for 15 minutes.

A 250 ml baffled flask containing 50 ml of Seed Medium was inoculated with Brevibacterium ammoniagenes ATCC 39101 and incubated at 31 300 RPM for 24 hours. Approximately 5.0 ml of the log phase culture (O.D.640 = 3.0) was sonicated to disaggregate clumps of cells. The sonicated cell suspension was diluted with sterile 0.9% NaCI and plated on Nutrient Agar (Difco) to obtain single colonies. The plates were incubated at 30"C for 72 hours.

A single colony from the plates was inoculated into a 250 ml baffled flask containing 50 ml Seed Medium and incubated at 310C, 300 RPM for 16 hours. At this time, the seed culture was in the early log phase of growth (O.D.640 = 1.7). The seed culture was used to inoculate 250 ml baffled flasks containing 50 ml Production Medium to an initial O.D640 of 0.05. To each flask, 50 l of 25.0% Pluronic 61 (BASF Wyandotte) antifoam was added. The production flasks were incubated at 31"C, 300 RPM. Analysis by HPLC of a sample of the broth harvested after 48 hours showed proline levels of 15.8 gm/l.

The microorganisms of this strain may be utilized to produce proline by any conventional aerobic method of cultivation or fermentation. Fermentation typically is carried out at 20 to 450C., preferably 28 to 35 C., and at a pH of 5 to 9. Calcium carbonate and ammonia may be employed for adjustment of the pH of the medium.

The culture medium may be any fermentation medium containing a carbon source, nitrogen source, inorganic salts and, where desired, other minor organic nutrients. As the carbon source, fermentable sugars, protein hydrolysates and proteins may be used. As the nitrogen source, urea, ammonium salts of organic acids (e.g., ammonium acetate or ammonium oxalate) and ammonium salts of inorganic acids (e.g., ammonium sulfate, ammonium nitrate or ammonium chloride) may be satisfactory. The amounts of the carbon and nitrogen sources in the medium are from 0.001 to 20 w/v percent. Inorganic elements (e.g., potassium phosphate or magnesium sulfate) and/or vitamins (e.g., thiamine) may be added as well.

Biotin, a vitamin B complex, is a necessary nutrient for cultivation of Brevibacterium ammoniagenes ATCC 39101. The concentration of biotin in the culture medium may be from about 50 to about 500 g/l.

Concentrations below this range tend to force this microorganism toward production of glutamic acid, which is an undesired contaminant when proline is the amino acid sought to be produced.

The addition of other organic nutrients to the culture medium may be desired. The microorganism of this invention is considered to be a bradytroph, that is, a slow grower which is more likely to thrive when grown with complex nutrients, than on a minimal medium. For example, organic nutrients (e.g., corn steep liquor, peptones, protein hydrolyzates or yeast extracts), inorganic elements (e.g., potassium phosphate or magnesium sulfate), andior vitamins (e.g., thiamine) may be added.

The preferred culture medium for microorganisms of this type should include organic nutrients. One of the preferred organic nutrients for flask-level fermentation is Bacto-Soytone (Difco), an enzymatic hydrolyzate of soybean meal, at a concentration of about .1 to about .5, preferably about .3, grams per liter of culture medium. Alternatively, yeast extracts, corn steep liquors or peptones from other sources may be used. In addition, the vitamin thiamine is a preferred nutrient.

The fermentation is accomplished in about 16 to 176 hours, typically 72 hours at the flask-level, during which time proline accumulates in the fermentation broth. Cells and other solid culture components may be removed from the broth by conventional procedures such as filtration or centrifugation.

Known procedures may be used in the recovery and/or purification of proline from the filtrate or supernatant solution. For instance, the filtered fermentation broth may be treated by using an ion-exchange resin. The proline may be crystallized from the resulting solution.

The principles, preferred embodiments and modes of operation of the present invention have been described in the foregoing specification. The invention which is intended to be protected herein, however, is not to be construed as limited to the particularforms disclosed, since these are to be regarded as illustrative rather than restrictive. Variations and changes may be made by those skilled in the art without departing from the spirit of the invention.

Claims (17)

1. An improved strain of an L-proline-producing microorganism, characterised by multiple proline analogue resistance and the capacity to overproduce L-proline.

2. The improved strain of claim 1, wherein the microorganism is resistant to two or more of the proline analogues 3,4-dehydro-DL-proline,azetidine-2-carboxylic acid, proline hydroxamate and D-proline.

3. The improved strain of claim 2, wherein the microorganism is a strain of Brevibacterium ammonia genes and is resistant to 3,4-dehydro-DL-proline and L-azetidine-2-carboxylic acid.

4. The improved strain of claim 3, wherein the microorganism is Brevibacterium ammonia genes ATCC 39101.

5. The improved strain of any one of claims 1 to 4, further characterised by utilizing a nutrient medium comprising carbon and nitrogen sources in the amounts of from about 0.001 to about 20.0 w/v percent.

6. The improved strain of claim 5, wherein said nutrient medium comprises biotin in the amount of about 50 to about 500 micrograms per liter.

7. The improved strain of claim 5 or 6 wherein said nutrient medium comprises at least one complex nutrient selected from peptones, protein hydrolyzates, yeast extracts and corn steep liquors.

8. A method for obtaining microorganisms capable of improved production of L-proline, comprising: (a) exposing L-proline producing microorganisms to a first proline analogue, (b) growing the first proline analogue-resistant colonies which survive the exposure of Step (a), (c) exposing said first proline analogue-resistant colonies to a second proline analogue, and (d) growing the first and second proline analogue-resistant colonies which survive the exposure of Step (c).

9. The method of claim 8, wherein said first proline analogue and said second proline analogue are different and each is 3,4-dehydro-DL-proline, azetidine-2-carboxylic acid, proline hydroxamate or D-proline.

10. The method of claim 8 substantially as hereinbefore described.

11. An improved strain of microorganism when obtained by the process of any of claims 8 to 10.

12. A method of producing L-proline, comprising: (a) selecting a strain of L-proline producing microorganisms which is resistant to multiple proline analogues, (b) cultivating or fermenting said strain in a culture medium comprising a carbon source and a nitrogen source, and (c) recovering L-proline from the culture medium.

13. The method of claim 12, wherein said strain of L-proline producing microorganisms is a strain of Brevibacterium ammoniagenes and is resistant to two different proline analogues selected from 3,4-dehvdro-DL-proline, L-azetidine-2-carboxylic acid, proline hydroxamate and D-proline.

14. The method of claim 13 in which said strain of L-proline-producing microorganisms is Brevibacterium ammonia genes ATCC 39101.

15. The method of claim 12, 13 or 14wherein the said culture medium comprises at least one complex nutrient selected from peptones, protein hydrolyzates, yeast extracts and corn steep liquors.

16. The method of claim 12 substantially as hereinbefore described.

17. L-proline when produced by the process of any of claims 12 to 16.